Method for diagnosing infectious diseases

ABSTRACT

A method for diagnosing the presence in an animal of an infection due to a microorganism in which a test sample obtained from the animal is exposed to a population of the microorganism and the presence of binding of the microorganism by an antibody in the test sample is determined.

This application is a continuation of U.S. patent application Ser. No.10/832,761, now U.S. Pat. No. 7,276,350, filed Apr. 27, 2004.

FIELD OF THE INVENTION

The invention pertains to the field of diagnosing infection due to amicrobial organism.

BACKGROUND OF THE INVENTION

Perhaps the most important aspect in diagnosing the cause of symptomsexperienced by a patient when an infectious agent is suspected as beingthe cause of the symptoms is the establishment of the identity of thespecific organism that is etiologically responsible for the symptoms.

Classically, the identity of an infectious microorganism has beenestablished by isolating the organism from the body of a patient,culturing the organism on a suitable culture medium, and identifying thecultured organism based on biochemical, immunological, or other tests.This method suffers from several disadvantages. Diagnosis by culture andidentification often requires a substantial period of time when growingorganisms that have a slow growth rate. For example, standard cultureand identification methods for Mycobacterium avium subsp.paratuberculosis may require 8 to 16 weeks or more to perform due to thevery slow growth rate of this organism. Another disadvantage to cultureand identification methods of diagnosis is that the particular organismcausing disease in a patient may fail to grow on standard culture media,leading to a negative culture result and a failure in diagnosis.Additionally, because such methods require the isolation of aninfectious organism from a patient, these methods are inappropriate attimes when the patient is not shedding the organism or if the organismis located in an inaccessible location within the body of the patient.

In recent years, molecular biological and immunological methods havebeen developed for the diagnosis of infectious diseases. These methodsgenerally fall into three categories, detection of genome nucleic acids,detection of protein, and detection of antibodies directed against apathogen.

Diagnosis by identification of genome nucleic acids is typicallyperformed using either or both amplification of DNA by polymerase chainreaction (PCR) followed by identification of PCR fragments produced orby use of probes that bind specifically to a portion of the genome of asuspected causative organism. These methods, especially when used incombination, can be very sensitive and specific methods to establish adiagnosis of a causative organism. There are several disadvantagesassociated with these methods. They are expensive, require sophisticatedtechnical expertise to perform, and generally take several days toobtain enough microorganisms for a diagnosis. Another significantdisadvantage associated with diagnosis by detection of genome nucleicacids is that an organism must be isolated in order to obtain the genomenucleic acids. Additionally, diagnosis based on DNA sequence may fail todistinguish between closely related microbial pathogens, such as betweendifferent strains of Mycobacterium, such as Mycobacterium avium subsp.paratuberculosis and Mycobacterium avium subsp. avium.

Diagnosis by identification of proteins is typically performed by anenzyme-linked immunosorbent assay (ELISA). In this test, an antigen froma test sample, typically a disrupted microorganism or a portion of amicroorganism, is captured by a first antibody that is specific for theantigen of interest and which is bound to a solid support. A labeledsecond antibody that binds to antibodies in test serum is then exposedto the solid support complex to provide a means for identification ofthe presence of the antigen. ELISA tests, however, suffer from severaldisadvantages including low sensitivity and the requirement to providetwo different antibodies for the detection of an antigen. ELISA testingrequires skilled laboratory technicians and can provide false results ifsamples are contaminated.

An example of an infectious disease for which currently availablediagnostic methods are inadequate is Johne's Disease, a disease incattle caused by Mycobacterium avium subsp. paratuberculosis (MAP).Johne's Disease results in decreased milk production and early cullingof infected cows resulting in an annual loss of approximately $1.5billion to the agricultural industry in the United States. Considerableevidence exists that MAP is also the causative organism of Crohn'sDisease in humans. Despite this significant impact on the U.S. economyand on human health, there is no effective diagnostic test to determineinfection by MAP.

At present, fecal culture is considered to be the most accurate means ofdiagnosing Johne's Disease. However, this diagnostic test has lowsensitivity (less than 50%) and is capable of detecting infections onlyin animals that are actively shedding MAP in their feces. Additionally,diagnosis of MAP by culture typically requires 8 to 16 weeks for growthof the organism.

Other diagnostic tests for Johne's Disease include PCR, complementfixation, agar gel immunodiffusion, and ELISA. These tests, each ofwhich utilizes a molecular extract of MAP, have inherently lowspecificity or sensitivity for MAP and suffer from the disadvantagespresent with these methods as indicated above.

A significant need exists for a diagnostic method that can be performedrapidly, is highly sensitive, is highly specific, and preferably can beperformed by an individual lacking sophisticated laboratory training.Particularly, a significant need exists for such a diagnostic methodthat is useful for diagnosing diseases such as those caused by MAP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the high specificity of the method of theinvention. Each bar represents the mean fluorescence intensity (mean+/−standard deviation of triplicate data) as determined by flowcytometry. FIG. 1 shows that high levels of antibody binding weredetected by fluorescent intensity on flow cytometry when serum from cowsinfected with MAP were mixed with MAP organisms. Flow cytometryfollowing mixing of samples with closely related mycobacterial speciesresulted in minimal antibody binding and a negative test result for MAPinfection.

FIG. 2 is a bar graph showing the high specificity of the method of theinvention. Each bar represents the mean fluorescence intensity (mean+/−standard deviation of triplicate data).

FIG. 3 is a bar graph showing the high sensitivity of the method of theinvention. Each bar represents the mean fluorescence intensity (mean+/−standard error of triplicate data) as determined by flow cytometry.FIG. 3 shows that serum from cows from farms determined to be MAP free(controls) binds only minimally to MAP organisms, serum from cowsdetermined to be MAP positive show high levels of antibody binding, andthat serum from cows from farms having MAP infection but which cows weredetermined by ELISA to be negative show levels of antibody bindinghigher than that of controls.

FIG. 4 is a bar graph showing the high sensitivity of the method of theinvention utilizing the simplified procedure of Examples 2 and 7. Eachbar represents the mean densimetric intensity (mean +/−standarddeviation of triplicate data) as determined by flow cytometry.

FIG. 5 is a bar graph showing results of the method of the invention fordiagnosing MAP infection using a dot blot procedure. Each bar representsthe mean fluorescence intensity. FIG. 5 also contains an insert showingimages of stained MAP organisms on dot blot. A: sera from cows from MAPnegative farm. B: sera from cows found to be MAP positive by IDEXX ELISA(IDEXX Laboratories, Inc., Westbrook, Me., USA).

DESCRIPTION OF THE INVENTION

In one embodiment, the invention is a method for diagnosing an infectionin an animal caused by a microorganism. The diagnostic method of theinvention is based on antibody binding to one or more specific antibodybinding sites that exist or existed on the surface of a particularmicroorganism. In accordance with this method, a test sample, preferablya serum sample, is obtained from an animal suspected of being infectedwith a microorganism. The test sample thus obtained is exposed to apopulation of the microorganism. It is then determined if the testsample contains an antibody that binds to the microorganism, preferablyto the surface of the microorganism. The test is positive for infectionwith the microorganism if antibodies in the test sample bind to themicroorganism.

The method of the invention is distinct from presently utilized methodsfor microbial diagnosis and provides several advantages that areunobtainable from such methods. Unlike culture methods, the method ofthe invention does not require isolation of an organism from an infectedanimal or the need to culture an organism in vitro. Therefore, themethod of the invention provides results in a much shorter time periodthan is achievable with culture methods and, in contrast to culturemethods, can provide a positive diagnosis even during times when themicroorganism is not able to be isolated from a host animal.

Unlike recent innovations in microbial diagnosis such as those based onnucleic acid or protein identification, the method of the invention isnot based on the determination of the presence of any specificmacromolecule peculiar to a particular organism. Also, unlike testsbased on antibody binding, such as ELISA testing, the method of theinvention does not present an antibody to determine if it binds to anextract of a microorganism or portion of a microorganism that is presentin a host animal. Rather, the method of the invention is based upondetermining that one or more antibodies present in a test sampleisolated from the body of a host animal binds to a particularmicroorganism that is brought into contact with the test sample.

Thus, the method of the invention provides several advantages previouslyunobtainable by present diagnostic methods. The method of the inventionmay be performed rapidly. In a field version of the method of theinvention, a positive or negative test result may be obtained rapidly,typically within about two hours. The method of the invention isextremely sensitive, more sensitive than presently available methods.The method of the invention can be used to provide a positive diagnosiseven during periods when a microbial pathogen is not detectable in, orisolatable from, a host animal. Additionally, the method of theinvention has a specificity that is higher than is obtained with otherpresently available methods of diagnosis.

The method of the invention is useful for the diagnosis of microbialinfections in animals. Such animals include mammals, such as humans andnon-human primates, carnivores such as dogs, cats, bears, and weasels,ungulate ruminants and non-ruminants such as horses, cattle, goats,sheep, pigs, non-ungulate ruminants such as camels and llamas,pinnipedia such as seals and sea lions, lagomorpha such as rabbits andhares, rodentia such as squirrels, rats, and mice, cetacea such aswhales, dolphins, and porpoises, and proboscidea such as elephants. Suchanimals also include non-mammalian vertebrates such as birds, reptiles,amphibians, and fish.

A suitable test sample that is obtained from an animal in accordancewith the method of the invention may be any fluid or tissue in which anantibody that specifically binds to a suspected causative organism wouldbe present if the animal were infected with that organism. Typically,the test sample is blood or a portion thereof, such as plasma orpreferably serum. However, it is contemplated that other sample sourcesmay be utilized in accordance with the invention. The selection of suchsource of test sample will vary depending, primarily, on the symptomsand signs of an infected animal and the suspected cause of such symptomsor signs. Thus, the test sample may be obtained from fluids such assaliva, milk, pus, tears and other ocular discharges, nasal discharges,sputum, cerebrospinal fluid, peritoneal or pleural fluid, urine, feces,and vaginal, uterine, or urethral secretions and discharges. Fluids mayalso include those that are produced as part of a pathologic processsuch as exudates or transudates, such as from the skin, the pleural orperitoneal cavity, the oral cavity, or from the digestive, respiratory,or genital system. The test sample may also be a solid tissue sample ifappropriate for diagnosis of a particular disease.

The test sample may be obtained by whatever method is appropriate toobtain such a sample. Thus, the test sample may be obtained by methodssuch as syringe withdrawal of fluid, including vascular puncture, suchas by venipuncture, or by withdrawal of fluid from other sources asdescribed above, or by biopsy.

The organism that is diagnosed by the method of the invention is anymicroorganism that is capable of eliciting an antibody response in ananimal infected by such microorganism. Thus, infective microorganismsthat may be diagnosed by the method of the invention include bacteria,fungi, viruses, protozoa, rickettsia, and chlamydia.

The invention is described in detail herein with reference tomycobacterial infections, and particularly with reference toMycobacterium avium, and most particularly with reference toMycobacterium avium subsp. paratuberculosis (MAP), the causativeorganism of Johne's Disease in cattle and Crohn's Disease in humans.This organism has proven to be a very difficult organism to establish asthe cause of disease symptoms in cattle and in people and presents,therefore, a significant test to establish the efficacy, specificity,and sensitivity of the method of the present invention.

The test sample may be exposed to a population of microorganism in anyway that permits antibodies that are contained in the test sample tointeract with the microorganisms. Thus, in a preferred embodiment, thetest sample and the microorganism are combined in a vessel such as atest tube or a well and are mixed together, such as by stirring ortapping the exterior of the test tube or well. The test sample and themicroorganism may also be reacted together on a surface such as on aslide, filter, or membrane, such as a nitrocellulose membrane.

In a preferred embodiment of the invention, the test sample is exposedto a population of intact whole microorganisms. In this way, antibodybinding sites on the entire surface of the microorganism are availablefor binding to antibodies in the test sample. It is preferred, if intactmicroorganisms are used, that the microorganisms be killed so as toavoid the risk of infection to humans and to other animals.

A preferred method for killing the microorganisms is by exposure of themicroorganisms to a chemical fixative. One preferred chemical fixativeis formaldehyde which, when used to kill MAP organisms, maintains theability of surface antibody binding sites of MAP to bind with antibodiesin serum from animals infected with the organism. A preferredconcentration of formaldehyde is about 1% to 10% v/v, with aconcentration of about 2% most preferred. Other chemical fixatives thatmay be used to kill microorganisms for use in the method of theinvention include non-coagulant fixatives such as acetone,glyceraldehydes, glutaraldehyde, and paraformaldehyde, and lesspreferred coagulant fixatives such as ethanol and mercuric chloride.Ethanol, in concentrations tested by the inventors (70% v/v), destroyedthe ability of surface antibody binding sites of MAP to bind withantibodies in serum from MAP infected animals. It is conceived thatcoagulant fixatives such as ethanol may be useful for killingmicroorganisms to be used in the method of the invention, especially ifused to diagnose infections by microorganisms other than MAP, or thatcertain concentrations of such fixatives may be suitable for killingmicroorganisms and may not render the killed microorganisms unsuitablefor the method of the invention. Because of this uncertainty concerningethanol, coagulant fixatives such as ethanol are less preferred.

The method of the invention may alternatively be performed by exposingthe test sample to a population of disrupted or partial microorganisms,such as microorganisms that have been fractionated, or to one or moreisolated antibody binding sites of the surface of a microorganism. Suchportions of microorganisms preferably contain a significant portion ofthe surface of the microorganism so as to present a variety of surfaceantibody binding sites to the antibodies in a test sample. Thus,according to the method of the invention, exposing a test sample to apopulation of microorganism includes exposing the test sample to intactmicroorganisms, to disrupted or partial organisms, or to one or moreisolated antibody binding sites of a microorganism.

Following the exposure of the test sample to the population ofmicroorganisms, it is then determined if the combination containsconjugates of antibodies from the test sample and microorganisms fromthe population. Any method that is suitable to detect the presence ofantibody binding to an antigen is suitable for the method of theinvention.

In one preferred embodiment, antibody-microorganism binding isdetermined by flow cytometry. Such flow cytometry determination may beperformed by analysis of a sample obtained by mixing a suspensioncontaining a serum sample and a population of microorganisms with alabeled anti-antibody, typically a fluorescein-labeled anti-antibody. Inanother preferred embodiment, antibody-microorganism binding isdetermined by blot analysis, such as dot blot or Western blot analysis.Such dot blot determination may be performed by mixing a suspensioncontaining a serum sample and a population of microorganisms with ananti-antibody which is labeled, such as with biotin or colloidal gold,spotting this mixture on a membrane, such as a nitrocellulose orpolyvinylidene fluoride (PVDF) membrane, and determining the presence oflabeled microorganism fixed on the membrane. As detailed below,diagnosis of infection with such methods is accurate, sensitive, andspecific. Determination of infection with methods such as dot blotanalysis permits diagnosis to be made by visual inspection and suchmethods are therefore capable of being performed by individuals who arenot technically trained in sophisticated laboratory techniques.

The invention is further illustrated in the following non-limitingexamples.

EXAMPLE 1 Technique for Serological Diagnostic Test Using Flow Cytometry

Two microliters of serum samples obtained from bovine subjects and 49microliters of phosphate-buffered saline-10% Superblock (PierceBiotechnology, Inc., Rockford, Ill. USA)-0.05% Tween (buffer A) wereadded to a population of whole organisms of Mycobacterium species (5microliters packed volume) to form a suspension of organisms. Thesuspension was incubated at room temperature for 1 hr, washed threetimes with 100 microliters buffer A by centrifugation at 5000 rpm for 10min, mixed with fluorescent-labeled rabbit anti-bovine IgG antibody (MPBiomedicals (formerly ICN Biomedicals), Irvine, Calif., USA) diluted1:50 in buffer A, incubated at room temperature for 1 hr, and washedtwice with buffer A by centrifugation at 5000 rpm for 10 min. One tenthof the volume of the treated organisms was resuspended in 1 ml of bufferA and fluorescence on 10,000 organisms was analyzed by using a flowcytometer (LSR II, BD Biosciences, San Jose, Calif., USA).

EXAMPLE 2 Alternative Technique for Serological Diagnostic Test by FlowCytometry

The method of the invention was performed utilizing a simplifiedalternative technique for serological diagnosis by flow cytometry. Thisalternative technique requires a shorter time than the techniquedescribed in Example 1 and does not require centrifugation.

One microliter of bovine serum, 5 microliters of fluorescent-labeledrabbit anti-bovine IgG antibody (1.5 mg/ml), and 44 microliters ofbuffer A were added to whole organisms of MAP (5 microliters packedvolume), and incubated at room temperature for 1 hr. One tenth of thetreated organisms were suspended in 1 ml buffer A and fluorescence on10,000 organisms was analyzed using a flow cytometer.

EXAMPLE 3 Serological Diagnostic Test Using a Dot Blot Technique

One microliter of bovine serum samples and 9 microliters of buffer Awere added to whole organisms of MAP (5 microliters packed volume), andincubated at room temperature for 1 hr. The organisms were washed threetimes with 100 microliters of buffer A by centrifugation at 5000 rpm for10 min, mixed with undiluted colloidal gold-labeled rabbit anti-bovineIgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove,Pa., USA), incubated at room temperature for 1 hr, and washed twice with100 microliters of buffer A by centrifugation at 5000 rpm for 10minutes. The treated organisms were resuspended in 100 microliters ofbuffer A and spotted on a PVDF membrane (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA) by using a dot blot apparatus (Bio-Rad). Imagesof the stained organisms on the dot blot were captured, and theirdensitometric intensities measured, using a gel documentation system(ChemiDoc XRS, Bio-Rad).

EXAMPLE 4 Specificity of the Method of Invention

Pooled and individual serum samples from cows known to be infected withMAP were analyzed as described in Example 1. Serum samples from cowsknown to be infected with MAP were mixed with organism populations thatwere one of 5 strains of Mycobacterium avium subsp. avium (MAA), onestrain of Mycobacterium scrofulaceum, or 4 strains of MAP, respectivelyprior to detection of binding by flow cytometry. Results are shown inFIG. 1.

As shown in FIG. 1, the method of the invention correctly identifiedinfection with MAP in all samples and showed a lack of false positivediagnoses as the method of the invention did not show binding whenbacterial populations closely related to MAP were used as the testorganism. This study establishes the high specificity of the method ofthe invention, which is capable of distinguishing between very closelyrelated organisms.

EXAMPLE 5 Specificity of the Method of the Invention

Serum samples from 8 cows known to be infected with MAP were pooled. Thepooled serum sample was tested as described in Example 1 by combiningindividual 2 microliter samples of the pooled serum sample with one of 8different strains of MAP or with MAA. Results are shown graphically inFIG. 2.

As shown in FIG. 2, minimal antibody binding as shown by fluorescentintensity was found for the sample that was mixed with MAA organisms. Incontrast, a very high level of antibody binding was determined for eachof the serum samples combined with MAP organisms. This study establishesthe high specificity of the method of the invention, which is capable ofdistinguishing between very closely related organisms, even between MAPand MAA, and specifically identified infection with MAP even whendifferent strains of MAP were used as the test microorganism.

EXAMPLE 6 Sensitivity of the Method of the Invention

The method of the invention utilizing whole MAP organisms, as describedin Example 1, was tested in comparison with an ELISA-based testHerdChek® Mycobacterium paratuberculosis Test Kits (Johne's Disease)(IDEXX Laboratories, Inc., Westbrook, Me., USA). The method of theinvention was performed according to the procedure of Example 1utilizing (1) sera from cows in farms that were determined to be free ofMAP infection (MAP Free, n=8), (2) sera from cows in farms with MAPinfection, which cows were determined by the IDEXX HerdChek® ELISA testto be MAP negative (MAP Negative, n=5), and (3) sera from cows that weredetermined by the IDEXX HerdChek® ELISA to be MAP positive (MAPPositive, n=14). Resultant data are shown in Table 1.

In Table 1, the mean (2429)+2 S.D. of MAP Free serum results (4164) wasused as a cut-off value to evaluate MAP infections of tested MAPNegative and MAP Positive cows. All (100%) of samples diagnosed aspositive by the IDEXX ELISA test were diagnosed as positive by themethod of the invention. Additionally, two of five (40%) of the samplesdetermined by the IDEXX ELISA test to be negative were diagnosed aspositive by the method of the invention.

TABLE 1 Serum Samples Fluorescent intensity of antibody binding MAP Free2085 2215 1250 4140 1701 2471 2794 2777 MAP Negative 4914 2411 6900 41203779 MAP Positive 16012 8487 7129 7468 20455 7719 13734 12344 2589315970 13392 8549 10497 17799 MAP Free (n = 8) - all samples found to benegative by ELISA testing were determined to be negative by the methodof the invention. MAP Negative (n = 5) - 2 of 5 samples (40%) positiveby method of invention. Results of samples found negative by ELISAtesting but positive by the method of the invention are underlined. MAPPositive (n = 14) - all samples found to be positive by ELISA testingwere determined to be positive by the method of the invention.

The data from this study is summarized in the bar graph of FIG. 3. InFIG. 3, the average antibody binding, as determined by fluorescentintensity, is shown for each of three types of samples, cows from MAPfree farms, cows from MAP positive farms but which have been determinedby IDEXX ELISA test to be MAP negative, and cows determined to bepositive for MAP by IDEXX ELISA test.

The data of Table 1 and FIG. 3 establish that the method of theinvention is more sensitive than that of the most commonly usednon-culture method presently in use for diagnosing Johne's Disease incattle. The IDEXX ELISA test had a false negative rate of 40% asestablished when later confirmed with the method of the invention, inwhich the false negative rate was 0%.

EXAMPLE 7 Alternative Technique for Diagnosis of MAP by Flow Cytometry

Sera from cows determined by IDEXX ELISA to be MAP negative were pooledand sera from cows determined by IDEXX ELISA to be MAP positive by IDEXXELISA test were pooled to provide two samples. The samples were testedfor the presence of MAP infection by the simplified, alternativetechnique described in Example 2. MAP organisms, pooled serum, andfluorescent-labeled secondary antibody were mixed, incubated for 1 hr,and antibody binding to MAP was determined by flow cytometry. Theresults were compared with a control sample which contained MAPorganisms and secondary antibody but no serum. Results are shown in thebar graph of FIG. 4.

As shown in FIG. 4, the control sample (None) showed a low level offluorescent activity as determined by flow cytometry. Additionally, theMAP positive serum showed a very high level of fluorescent activityindicating a high level of serum antibody binding with the MAPorganisms. Unexpectedly, the fluorescent activity of the MAP free pooledsera, that found to be negative by ELISA test, showed a level offluorescent activity about twice that of control. This result indicatesthat the ELISA test incorrectly identified MAP infected cows as beingfree of infection. The diagnostic test according to the invention,however, correctly identified that antibodies due to MAP infection werepresent in the pooled serum from this group.

The results of this study establish that, even with a simplifiedprocedure requiring a shortened processing time and no centrifugation,the method of the invention is more sensitive than the most commonlyused present method for diagnosis of MAP infection in cattle.Additionally, because portable flow cytometers are commerciallyavailable, the method of the invention is useful for field, onsitediagnosis.

EXAMPLE 8 Diagnosis of MAP by Dot Blot Technique

Sera from cows found to be MAP negative by ELISA test were pooled andsera from cows found to be MAP positive by ELISA test were pooled toprovide two samples. The samples were treated as described above inExample 3 and diagnosed by dot blot technique. The results are shown inFIG. 5.

As shown in FIG. 5, the dot blot technique of the invention showed ahigh degree of antibody binding in sera from cows previously found to beMAP positive by ELISA test. Additionally, antibody binding to MAP wasdetected by this method even in sera from cows previously found to benegative by ELISA testing, establishing the high sensitivity of themethod of the invention as performed by dot blot technique.

The dot blot technique is a simple method that can be performed easilyin the laboratory by trained personnel. Additionally, because results ofdot blot technique are evaluated visually, this technique can beperformed in field conditions even by individuals not trained inlaboratory techniques. Thus, the method of the invention is useful forboth laboratory and field diagnosis of MAP infected individuals, andsuch diagnosis may be performed by either technical or non-technicalpersonnel.

Further modifications, uses, and applications of the invention describedherein will be apparent to those skilled in the art. It is intended thatsuch modifications be encompassed in the claims that follow.

1. A method for diagnosing the presence in an animal of an infectioncaused by a microorganism comprising obtaining a test sample from theanimal, exposing the test sample to a population of the microorganismfor a time sufficient to permit an antibody in the test sample thatbinds to an antibody binding site of the microorganism to bind to theantibody binding site, then putting the microorganisms that were exposedto the test sample in suspension, exposing the test sample and thepopulation of the microorganisms to a labeled anti-antibody before,during, or after the microorganisms that were exposed to the test sampleare put in suspension, determining a quantitative level of binding ofthe suspended microorganisms to antibodies in the test sample bydetecting an absolute level of the label that is bound to themicroorganisms, and comparing the level of the antibody binding in thetest sample with the level of antibody binding from a negative control,wherein the presence of infection is diagnosed if the level of antibodybinding in the test sample is higher than that in the control.
 2. Themethod of claim 1 wherein the animal is a mammal.
 3. The method of claim2 wherein the mammal is a bovine.
 4. The method of claim 1 wherein themicroorganism is a bacterium.
 5. The method of claim 4 wherein thebacterium is a mycobacterium.
 6. The method of claim 5 wherein themycobacterium is Mycobacterium avium subsp. parcatuberculosis.
 7. Themethod of claim 1 wherein the test sample is serum.
 8. The method ofclaim 7 wherein the serum test sample is obtained from the animal byvenipuncture.
 9. The method of claim 1 wherein the test sample is afluid or bodily discharge selected from the group consisting of saliva,milk, pus, ocular discharge, nasal discharge, sputum, cerebrospinalfluid, peritoneal fluid, pleural fluid, urine, feces, vaginal secretionor discharge, uterine secretion or discharge, urethral secretion ordischarge, extidate, and transudate.
 10. The method of claim 1 whereinthe population of the microorganism contains intact wholemicroorganisms.
 11. The method of claim 10 wherein the intact wholemicroorganisms are killed microorganisms.
 12. The method of claim 1wherein the detection of the level of binding is by blot analysis.
 13. Amethod for diagnosing infection caused by Mycobacterium avium subsp.paratuberculosis (MAP) in an animal comprising obtaining a test samplefront the animal, exposing the test sample to a population of MAPorganisms for a time sufficient to permit an antibody in the test samplethat binds to an antibody binding site of MAP to bind to the antibodybinding site, then putting the MAP microorganisms that were exposed tothe test sample in suspension, exposing the test sample and thepopulation of the MAP microorganisms to a labeled anti-antibody before,during, or after the MAP microorganisms that were exposed to the testsample are put in suspension, determining a quantitative level ofbinding of the MAP organisms to antibodies in the test sample bydetecting an absolute level of the label that is bound to themicroorganisms, and comparing the level of the antibody binding in thetest sample with the level of antibody binding from a negative control,wherein the presence of MAP infection is diagnosed if the level ofantibody binding in the test sample is higher than that in the control.14. The method of claim 13 wherein the infection is Johne's Disease. 15.The method of claim 13 wherein the animal is a bovine.
 16. The method ofclaim 13 wherein the test sample is serum.
 17. The method of claim 13wherein intact whole MAP organisms are exposed to the test sample. 18.The method of claim 17 wherein the intact whale MAP organisms arekilled.
 19. The method of claim 13 wherein the detection of the level ofbinding is by blot analysis.